The current COVID-19 pandemic is a worldwide, rapidly developing, health crisis caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). As of May 18, 2020, over 4.7 million infections are confirmed globally and over 315,000 people have died from COVID-19 related complications. Efforts to develop and test COVID-19 vaccines are in high gear. In the meantime, there is a dire need for fast and robust in-vitro tests that can be used to study the mechanisms of host-virus interactions and help assess whether existing antivirals could be used against for SARS-CoV-2. Current static 2D cell culture systems and animal-based models are of limited use for these purposes. To address this gap, the proposed project aims to develop organ- on-chip (OOC)-based assays for quantifying SARS-CoV-2 inoculation and replication in three human tissues that have been shown to be severely affected by SARS-CoV-2. In order to enable an immediate start, a fast timeline, and milestones with translational impact, the approach of this supplement will mainly repurpose already existing, validated, and commercialized OOC models that were developed under the parent grant. AIM1 is to develop SARS-CoV-2 assays for kidney proximal tubule and vascular endothelium, models that were initially developed for assessing drug toxicity and drug transport. In addition, an OOC model of the lung alveolus will be developed. SARS-CoV-2 Wuhan Reference Strain, the SARS-CoV-2 Spike Mutation D614G Strain, as well as a Spike-pseudotyped lentivirus will be tested and compared for differences in inoculation rate and replication rate (AIM2). The assay protocols will include introducing the viruses via the perfusate to the lumen of the tissue structures in order to bring the virus in contact with the ACE2 and CD 147 receptors that reside on the apical side of the cell and are responsible for virus binding and subsequent endocytosis. To quantify viral inoculation, the tissues will be removed after a short but adequate incubation period. The viruses will be extracted from the tissues, serially diluted and quantified using plaque assays. In order to assess viral replication, tissues will be harvested from the chips after a pre-determined, longer, incubation period that gives the cells enough time for viral replication. Viral load will be quantified with plaque assays. AIM3 is to use the OOC-based assays for testing a number of candidate antivirals and compare their effect against baseline SARS-COVID-19 virus load. The list of antivirals to be tested includes antibodies against ACE2 and CD147 receptors; RNA polymerase inhibitor Remdesivir; PAMP RNA, a RIG-agonist and interferon inducer; and the antimalarial chloroquine. The data obtained from the OOC assays will be correlated with pre-existing in-vitro data, animal data, and clinical findings. The technology can be quickly made available to the research community. Models of other tissue structures affected by SARS-COVID-19, such as myocardium, intestinal mucosa, and kidney glomerulus can be subsequently added to the portfolio. Further, the models can be tailored to include cells from elderly patients or mimic conditions connected with severe outcomes, such as diabetes, hypertension, or kidney disease.